The mammalian sympathetic nervous system is composed of various cellular tissues having the unique capability of synthesizing postsynaptic neurotransmitter compounds. These compounds are biogenic amines termed catecholamines, a term derived from the catechol, or dihydroxybenzene, nucleus common to each of the compounds. These catecholamines include dopamine, norepinephrine and epinephrine.
Aberrations of the sympathetic nervous system can lead to a wide variety of adverse clinical manifestations. Therefore, accurate and reliable methods for quantifying the concentration of catecholamines in the body are critical to provide adequate monitoring of the system. Further, since these compounds are present in the body in very small amounts, the methods must be highly sensitive, that is, capable of detecting the compounds in very small amounts. The frequency at which these methods are conducted mandate further that they be highly reproducible under laboratory conditions when employing a variety of body tissues and fluids and provide the results quickly in order to facilitate diagnosis.
Radioenzymatic assays are sensitive analytical methods which have found wide use in the quantification of various biogenic amines. These assays are based on the enzymatic methylation of a specified compound to a radiolabeled product by an appropriate enzyme employing radioactive S-adenosylmethionine as the methyl donor. Most of the currently employed radioenzymatic assays lack the sensitivity necessary to quantify catecholamines in important biological samples, such as human plasma.
Henry et al. in Life Sciences 16:375 (1975) describe a useful radioenzymatic assay for specifically measuring norepinephrine in tissues, plasma and urine. This method relates to the conversion of norepinephrine to radiolabeled epinephrine employing partially purified bovine adrenal phenylethanolamine N-methyltransferase and tritiated S-adenosylmethionine.
Since the first disclosure of the phenylethanolamine N-methyltransferase based norepinephrine radioenzymatic assay described above, research has continued in an effort to improve the assay in order to provide maximal sensitivity and specificity. It has been determined that the enzymatic conversion of norepinephrine to epinephrine is severely inhibited by endogenous substances contained in partially purified phenylethanolamine N-methyltransferase enzyme preparations.
Known procedures for partially purifying phenylethanolamine N-methyltransferase exist but are incapable of removing contaminating methyltransferase enzymes and other substances. Competing methyltransferases of particular importance due to their high activity and widespread distribution in mammalian tissues include the protein methyltransferases and sulfhydryl methyltransferase. Further, sulfhydryl reducing agents such as dithiothreitol have been employed in radioenzymatic assays as antioxidants for catecholamines. Many of these compounds are substrates for sulfhydryl methyltransferases.
The use of phenylethanolamine N-methyltransferase contaminated with other methyltransferases reduces the sensitivity of the assay by reducing the rate of conversion of substrate to product and by increasing assay blanks. The amount of radiolabeled epinephrine produced is reduced since the contaminant methyltransferases exhaust the methyl donor S-adenosylmethionine and generate S-adenosylhomocysteine, a known potent methyltransferase inhibitor.
Several major problems exist in isolating phenylethanolamine N-methyltransferase in sufficient purity to be suitable for use in radioenzymatic assays. The first problem is that the enzyme exists in multiple charge and molecular weight forms. Secondly, various catecholamines copurify with other large molecular weight substances such as phenylethanolamine N-methyltransferase.
The present invention overcomes these problems and provides a process for purifying phenylethanolamine N-methyltransferase in high yield suitable for use in the analysis of a number of biogenic amines. More specifically, this process can be conducted under standard laboratory conditions within a comparatively short time to provide purified phenylethanolamine N-methyltransferase capable of providing maximal conversion of norepinephrine to tritiated epinephrine.